Method of inducing autophagy and activating toll-like receptor
A method of inducing autophagy in a cell is achieved by contacting the cell with graphene oxide (GO) in an amount effective to induce autophagy in the cell, wherein the cell expresses at least one of TLR-4 (Toll-like receptor 4) and TLR-9 (Toll-like receptor 9). Differences between autophagy triggered by GO and other conventional agonists such as rapamycin have been observed. GO may activate autophagy in some cells that may not be triggered by rapamycin. The cell reveals no apparent apoptosis after treatment of the graphene oxide. A method of activating a Toll-like receptor in a cell is also herein provided.
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- PHOTORESIST AND FORMATION METHOD THEREOF
- PHOTORESIST AND FORMATION METHOD THEREOF
This application is a Divisional of co-pending application Ser. No. 13/913,716, filed on Jun. 10, 2013, for which priority is claimed under 35 U.S.C. §120, the entire contents of all of which are hereby incorporated by reference.
BACKGROUND OF THE INVENTION1. Field of the Invention
The present invention relates to a method of inducing autophagy in a cell, particularly to a method of inducing autophagy in a cell by activating Toll-like receptors.
2. Description of the Prior Art
Graphene and its oxidized form, graphene oxide (GO), have drawn intense attention in recent years for biological and medical applications. The surface of GO contains hydrophilic oxygen-containing functional groups (i.e. hydroxyl, epoxyl and carboxyl tails) on the basal plane and edges, rendering GO amenable to stable dispersion in water and functionalization. These attributes have prompted the use of GO for bioimaging, cellular probing, cellular growth and differentiation, gene and drug delivery and photothermal therapy. These burgeoning applications in biomedicine entail the need to evaluate the in vitro and in vivo safety of GO.
Autophagy is a process that degrades intracellular components in response to stressful conditions (e.g. starvation and infection) and is linked to cellular processes as diverse as cell survival, cell death, pathogen clearance and antigen presentation. Autophagy involves the formation of double-membraned vesicles termed autophagosomes, which sequester cytoplasm and organelles and then fuse with lysosomes to form autolysosomes, thus degrading the contents of the vacuole. Autophagy is negatively controlled by mTOR (mammalian target of rapamycin) complex 1 (mTORC1) and inhibition of mTORC1 kinase activity initiates the formation of autophagosome that comprises a complex consisting of Beclin 1 and other factors. The autophagosome formation also involves the conversion of microtubule-associated protein light chain 3 (LC3-I) to the lipidated form LC3-II, consequently conversion from LC3-I to LC3-II is a common indicator of autophagy.
Toll-like receptors (TLRs) are important receptors for the detection of microbial antigens and subsequent induction of innate immune responses. Among the TLRs, TLR2 recognizes bacterial lipoproteins while TLR3 detects virus-derived dsRNA. TLR4 recognizes lipopolysaccharides (LPS) and TLR5 recognizes bacterial flagellin. TLR7 mediates recognition of viral ssRNA while TLR9 senses unmethylated DNA with CpG motifs derived from bacteria and viruses. Upon engagement with cognate ligands, the TLRs transduce signals by first recruiting adaptor proteins including myeloid differentiating factor 88 (MyD88) and TIR domain-containing adaptor inducing IFN-beta (TRIF), followed by activation of downstream signaling proteins such as TRAF6 and NF-κB, eventually resulting in various cellular responses including secretion of cytokines and interferons (IFNs).
The connection between autophagy and TLRs was discovered in 2007 as it was found that TLRs signaling in macrophages links the autophagy pathway to phagocytosis and TLR4 stimulation enhances the autophagic elimination of phagocytosed mycobacteria in macrophages. Ensuing studies further reported that TLR2, TLR3 and TLR7 play roles in autophagy induction. To date the precise mechanisms regulating the TLRs-elicited autophagy remain to be established although agonists stimulating TLR2, TLR3, TLR4 and TLR7 were shown to trigger autophagy.
SUMMARY OF THE INVENTIONThe present invention is directed to provide a new mechanism by which cells respond to nanomaterials and underscores the importance of future safety evaluation of nanomaterials.
According to an embodiment, A method of inducing autophagy in a cell is achieved by contacting the cell with graphene oxide (GO) in an amount effective to induce autophagy in the cell, wherein the cell expresses at least one of TLR-4 (Toll-like receptor 4) and TLR-9 (Toll-like receptor 9).
According to another embodiment, a method of activating a Toll-like receptor in a cell is achieved by contacting the cell with graphene oxide in an amount effective to activate a at least one of TLR-2 (Toll-like receptor 2), TLR-4 (Toll-like receptor 4), TLR-7 (Toll-like receptor 7) and TLR-9 (Toll-like receptor 9) in the cell, whereby at least one of TLR-2, TLR-4, TLR-7 and TLR-9 are activated in the cell.
Other advantages of the present invention will become apparent from the following descriptions taken in conjunction with the accompanying drawings wherein certain embodiments of the present invention are set forth by way of illustration and examples.
The foregoing aspects and many of the accompanying advantages of this invention will become more readily appreciated as the same becomes better understood by reference to the following detailed descriptions, when taken in conjunction with the accompanying drawings, wherein:
The present invention demonstrated that treatment of cells with GO simultaneously triggers autophagy and mainly TLR4/TLR9-regulated inflammatory responses.
In one embodiment, the particle sizes of the graphene oxide may range from 100 nm to 3 μm, preferably 100 nm to 800 nm and average around 450 nm. The concentration of the graphene oxide may be greater than or equal to 5 μM, preferably greater than or equal to 50 μM or 100 μM.
Autophagy triggered by GO have been observed in various types of cells such as cancer cells and immune cells. In one preferred embodiment, cancer cells may include an ovarian cancer cell (SKOV3), a brain can cell (ALTS1C1), a prostate cancer cell (Tramp C1), a cervical cancer cell (HeLa), a lung cancer cell (A549), a liver cancer cell (Mahlavu) or a colon cancer cell (CT26). Immune cells may include primary immune cells such as macrophages.
In addition, differences between autophagy triggered by GO and other conventional agonists such as rapamycin have been observed. GO may activate autophagy in some cells that may not be triggered by rapamycin. Some cells are likely damaged by rapamycin in comparison to GO treatment. The cell reveals no apparent apoptosis or necrosis after treatment of the graphene oxide. Furthermore, the autophagy induced by GO may be more than 40% of the cell. In one preferred embodiment, autophagy may be induced in essentially 80% or more of the cell.
The autophagy presented by the present invention was at least partly regulated by the TLRs pathway. Very importantly, TLRs are well known detectors for various biological molecules, but their sensing of non-living nanomaterials such as GO has yet to be reported. Neither has any study documented that nanomaterials can induce autophagy via the regulation of TLRs. This present invention thus presents a new mechanism by which cells respond to nanomaterials and underscores the importance of future safety evaluation of nanomaterials.
The detailed explanation of the present invention is described as follows. The described preferred embodiments are presented for purposes of illustrations and description, and they are not intended to limit the scope of the present invention.
Reference of Chen et al. (Biomaterials 33 (2012) 6559-6569, hence abbreviated as Reference) is herein incorporated by reference in its entirety.
Preparation and Characterization of GO
Large GO with a size of ≈2.4 μm was prepared from natural graphite (Bay Carbon, SP-1, average particle size ≈30 μm) by the modified Hummers method as described previously [20] and dispersed in water. The solution was centrifuged (7,200×g for 5 min) to remove unexfoliated GO and byproducts and centrifuged again (400×g for 15 min) to remove broken fragments and debris. The pellet was dried under vacuum overnight to yield the large GO, weighed on a Sartorius SE2 ultra-micro balance with 0.1 μg resolution and dissolved in deionized water to a final concentration of 250 μg/ml. Small GO with a size of ≈350 μm was prepared via tip sonication (Misonix Sonicator 3000) of the large GO solution in an ice bath at a power of 30 W for 1 h, filtered through a 0.45 μm syringe filter (Sartorius Stedim Biotech) and dried under vacuum overnight. The small GO was weighed and dissolved in water to a desired concentration.
The surface morphology of GO was characterized with an atomic force microscope (AFM, XE-70, Park System) in tapping mode using the aluminum coating silicon probe (frequency 300 kHz, spring constants 40 N/m, scanning rate 1 Hz), under ambient conditions and scanning line of 512. High-resolution X-ray photoelectron spectroscopy (HRXPS) and attenuated total reflectance HRXPS were performed on a Kratos Axis Ultra DLD using a focused monochromatic A1 X-ray source (1486.6 eV). The Fourier transform infrared (ATR-FTIR) spectra of GO were recorded using a Perkin-Elmer Spectrum RXI FTIR spectrometer with 2 cm−1 resolution and 32 scans, and the background was collected in the absence of samples. The size distribution of GO was characterized by using Dynamic Light Scattering (380 ZLS, Nicomp, USA) from Particle Sizing Systems at room temperature.
Cell Culture and Treatment with GO
The mouse macrophage cell line RAW264.7 was maintained in Dulbecco's modified Eagles medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco) and subcultured upon 70-80% confluency. For GO treatment, the cells were seeded to 6-well plates (3×105 cells/cm2) overnight and cultured using the medium supplemented with GO at final concentrations of 5 or 100 μg/ml for 24 h. In parallel, the cells were treated with LPS (10 μg/ml, Sigma) for 24 h as the positive control. After the treatment, the cell morphology and vacuoles were observed under the phase contrast microscope.
Transmission Electron Microscopy (TEM)
The cells were harvested, centrifuged (215×g, 10 min), washed with cold PBS and fixed with 2.5% glutaraldehyde (in 0.2 M sodium cacodylate, pH 7.4). The samples were then fixed in 1% OsO4 for 1 h at 4° C., dehydrated with increasing concentrations of ethanol, embedded in spur resin and sectioned. The ultrathin sections were stained with uranyl acetate and observed under the TEM.
Immunofluorescence Microscopy
The cells were fixed and permeabilized as described previously [4], followed by extensive washing and primary antibody staining (1:100 dilution) for 1 h at 4° C. in the dark. The primary antibody was specific for LC3 (4108, Cell Signaling Technology), Beclin 1 (ab55878, Abcam), TLR4 (14-9924, eBioscience), TLR9 (ab17236, abcam), MyD88 (ab2068, abcam), TRAF6 (ab33915, abcam), phosphorylated NF-κB (3033, Cell Signaling Technology) or IRF3 (sc-15991, Santa Cruz Biotechnology). After washing, the cells were incubated with the goat anti-mouse antibody conjugated with Alexa 488 (for TLR9, Invitrogen), goat anti-rabbit antibody conjugated with Alexa 488 (for LC3, MyD88, TRAF6 and NF-κB, Invitrogen) or donkey anti-goat IgG conjugated with Dylight 488 (for IRF3, Jackson ImmunoResearch) for 1 h at 4° C. in the dark. After washing, the cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Vector Labs) and visualized with a confocal microscope (Nikon TE2000 equipped with the confocal upgrade laser kit). Fifty to one hundred cells in the images for LC3 were counted for quantification of LC3+ cells.
ELISA and Western Blot
At 24 h post-treatment, the supernatant was collected from the GO-treated cell culture and analyzed using ELISA kits specific for mouse IL-2, IL-10, TNF-α, IFN-β and IFN-γ. The cells were lysed for Western blot using primary antibodies (1:1000 dilution) specific for LC3, Beclin 1 or β-actin (A-2066, Sigma) and the secondary antibody was HRP-conjugated IgG (1:5000 dilution, Amersham Biosciences). The images were developed using the GeneGnome HR scanner (Syngene).
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted from the cells using the NucleoSpin® RNA II purification kit (Clontech) and reverse transcribed to cDNA using the MMLV Reverse Transcriptase 1 st-Strand cDNA Synthesis Kit (Epicentre Biotechnologies). The RT-PCR reactions were performed using Taq DNA polymerase (Promega) in the Px2 Thermal Cycler (Thermo Electron) under the condition of 30 s at 95° C., 45 s at 60° C. and 30 s at 72° C., and the amplicons were subjected to 2% agarose gel electrophoresis. For TLRs transcription analysis, the cDNA was amplified using the Murine TLR RT-Primers (Invivogen).
Flow Cytometry
The cells were fixed and permeabilized with 4% formaldehyde and 0.5% Tween-20. After washing, the cells were incubated with the primary antibody (1:100 dilution) for 1 h at 4° C. in the dark. For TLR2 and TLR4 detection, the primary antibody was Alexa 488-conjugated MAb specific for mouse TLR2 (53-9024, eBioscience) or PE-conjugated MAb specific for mouse TLR4 (12-9924, eBioscience). For TLR7, TLR9, MyD88 and TRAF6 detection, the cells were incubated with the primary antibody specific for mouse TLR7 (ab45371, abcam), TLR9 (ab17236, abcam), MyD88 (ab2068, Abcam) or TRAF6 (ab33915, Abcam) and then incubated with Alexa 488-conjugated goat anti-rabbit (for TLR7, MyD88 and TRAF6) or goat anti-mouse (for TLR9) IgG for 1 h at 4° C. in the dark. After washing, the cells were collected for flow cytometry (FACSCalibur, BD Biosciences) analyses.
Gene Knockdown by Small Interfering RNA (siRNA)
To knockdown specific genes, macrophages cells were transfected with 5 μg of scramble siRNA (SC-36869, Santa Cruz Biotechnology) or siRNA specific for TLR4, TLR9, MyD88, TRAF6 or TRIF (Santa Cruz Biotechnology). At 48 h post-transfection, cells were treated with GO or LPS as described above. The supernatant was collected 24 h later for ELISA and the cells were harvested for immunofluorescence microscopy and Western blot. Statistical analysis
All data represented the mean±standard deviation of at least 3 independent culture experiments. The data were statistically analyzed by one-way ANOVA. p<0.05 was considered significant.
Example 1 Preparation and Characterization of Large and Small GO NanosheetsLarge-size GO was prepared from natural graphite by the modified Hummers method while small-size GO was obtained by sonicating large GO into smaller pieces via tip sonication. Atomic force microscopy (AFM) images showed significant difference of lateral dimensions between large and small GO (
To examine how GO nanosheets influenced the macrophage, RAW264.7 cells were incubated with small GO at either 5 μg/ml (designated as GO5 group) or 100 μg/ml (designated as GO100 group) for 24 h. In comparison with the untreated control, GO5 induced the formation of small vacuoles inside the cells at 24 h post-incubation (
Since the GO-induced vacuoles were observed in cells treated with LPS, a ligand that induces both autophagy and TLR pathway, we surmised that GO triggered autophagy. Indeed, the transmission electron microscopy demonstrated that GO5 evoked the appearance of some autophagic vacuoles (AV) while GO100 and LPS triggered more prominent AV (
Beclin 1 and LC3 are two key proteins associated with the autophagy pathway and are common indicators of autophagy induction. LC3 is normally present diffusely in the cytosol but upon autophagy is converted from LC3-I (18 kD) to LC3-II (16 kD), accumulates on the autophagosome membrane and appears as dots. Immunofluorescence microscopy for Beclin 1 and LC3 (
GO Treatment of Macrophage Elicited the Cytokine Expression and TLR4/TLR9 Signaling
Since the interplay between autophagy and TLRs signaling was recently revealed, we were inspired to explore whether GO elicited TLRs-associated inflammatory responses. ELISA analysis (
Conversely, GO5 and GO100 evidently upregulated the transcription of TLR9 but barely triggered other TLRs genes, as depicted by RT-PCR analyses (
Since only the expression of TLR4 and TLR9 was markedly elicited by GO, we next examined the roles of TLR4 and TLR9 pathways on the inflammatory response. The TLR4 pathway signals through either TRIF or MyD88. The TRIF-dependent pathway results in activation and nuclear translocation of IRF3, thereby triggering the secretion of IFN-α/β. However, GO5 and GO100 neither evoked nuclear translocation of IRF3 (FIG. S5 of reference) nor elicited IFN-β expression (
Conversely, TLR4 signaling through MyD88 leads to the formation of MyD88/IRAK4/TRAF6 signalsome, nuclear translocation of phosphorylated NF-κB and subsequent cytokine expression. TLR9 stimulation recruits MyD88 and results in the formation of MyD88/IRAK4/TRAF6/TRAF3 complex, which relays signals either through IRF7 for IFN-α/β secretion, or through NF-κB for cytokine expression. As demonstrated by the flow cytometry (
Inhibition of TLR4/TLR9 Pathways Mitigated the GO-Induced Cytokine Response and Autophagy
To confirm the roles of individual signaling mediators on the cytokine response, the macrophage cells were treated with siRNA specific for TLR4, TLR9, MyD88, TRIF or TRAF6. Following the silencing as confirmed by RT-PCR (
Immunofluorescence microscopy (
To assess the responsiveness of cancer cells to GO, cells of different cancer types including human ovarian carcinoma (SKOV-3), murine astrocytoma (ALTS1C1), murine colon carcinoma (CT26) and murine prostate adenocarcinoma (TRAMP-C1) were cultured in medium supplemented with GO nanosheets (thickness<2 nm, lateral size ≈450 nm in mean diameter). Immunofluorescence microscopy revealed that GO at a concentration of 50 μg/ml (GO50 group) only induced evident autophagy in CT26 cells after 18 h (
The GO-induced autophagy was also dose-dependent in CT26 cells (
GO Activates TLR-4/9 Pathways in CT26 Cells.
Owing to the findings that GO provokes both TLR-4 and TLR-9 signaling pathways in macrophage cells in vitro, we surmised that GO also triggered TLR-4/9 cascades and their downstream cytokine (e.g. TNF-α and IL-1β) production in CT26 cells. Indeed, GO50 significantly provoked the production of TNF-α and IL-1β when compared with the untreated cells (
GO was phagocytosed by CT26 cells in a way related to TLR-4/9 signaling.
TLR-4 is a receptor on the cell surface whereas TLR-9 is produced in ER and translocates to endosome. To explore how GO entered the cells to engage TLR-9, we treated CT26 cells with FITC-conjugated beads as a marker of phagocytosis. Comparison of the cells treated with beads only with the cells co-treated with beads and GO50 (
GO-induced TLR-4/9 cascades were independent of the mTOR pathway.
mTOR is a negative autophagy regulator, and repressing the phosphorylation of mTOR and its upstream Akt can elicit autophagy. Indeed, rapamycin suppressed the phosphorylation of Akt and mTOR (
GO-induced TLR4/9 signaling was upstream of the GO-induced autophagy.
The interplay between autophagy and immunity has drawn intensive attention in recent years. It was shown that TLR-4 signaling can activate autophagy in a way dependent on ATG5 and Beclin 1. Oppositely, it was also suggested that autophagy regulates the activation of TLRs pathways. To elucidate the crosstalk between the GO-induced TLR-4/9 signaling and autophagy, CT26 cells were transfected with siTLR-4 or siTLR-9, followed by GO50 treatment. Compared with the scrambled siRNA, siTLR-4 and siTLR-9 significantly mitigated the GO-induced activation of LC3 and Beclin 1 (
To evaluate whether the opposite was true, we transfected cells with siRNA for atg5 and atg7 (genes essential for autophagy induction) to knockdown the GO-induced autophagy (
Beclin 1 is inactivated by the inhibitory interaction with TAB2/3, Bcl-2 and Bcl-xL in the usual state and TLR signaling can release Beclin 1 from the inhibitory molecules, enhance the interaction between Beclin 1 and MyD88, while activated TRAF6 stimulates Beclin 1 to initiate autophagy. Since GO50 induced TLR-4/9 and downstream signaling effectors MyD88 and TRAF6, and concurrently activated LC3, Beclin 1 and the ensuing autophagy, we propose that GO engagement of TLR-4/9 activates MyD88/TRAF6 and induces autophagy through the activation of Beclin 1 and LC3, in a way independent of the mTOR pathway.
GO Injection Suppressed Tumor Formation, Enhanced Cell Death, Autophagy and Immune Cell Infiltration
To assess the potential of GO-induced autophagy in cancer therapy, CT26 cells were injected subcutaneously into BALB/c mice, followed by intratumoral injections of PBS or GO at day 0 (when the tumor volume reached ≈30-40 mm3) and day 8. In comparison with PBS, GO alone significantly suppressed the tumor progression (
While the invention can be subject to various modifications and alternative forms, a specific example thereof has been shown in the drawings and is herein described in detail. It should be understood, however, that the invention is not to be limited to the particular form disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the appended claims.
Claims
1. A method of activating a Toll-like receptor in a cell, comprising:
- contacting the cell with graphene oxide in an amount effective to activate at least one of TLR-2 (Toll-like receptor 2), TLR-4 (Toll-like receptor 4), TLR-7 (Toll-like receptor 7) and TLR-9 (Toll-like receptor 9) in the cell, whereby at least one of TLR-2, TLR-4, TLR-7 and TLR-9 are activated in the cell.
2. The method as claimed in claim 1, wherein the cell is an immune cell.
3. The method as claimed in claim 1, wherein TLR-4 and TLR-9 are both activated in the cell.
4. The method as claimed in claim 1, wherein the particle sizes of the graphene oxide range from 100 nm to 3 μm.
5. The method as claimed in claim 1, wherein the particle sizes of the graphene oxide range from 100-800 nm.
6. The method as claimed in claim 1, wherein the concentration of the graphene oxide is greater than or equal to 5 μM.
7. The method as claimed in claim 1, wherein the concentration of the graphene oxide is greater than or equal to 100 μM.
8. The method as claimed in claim 1, wherein the cell reveals no apparent apoptosis or necrosis after treatment with the graphene oxide.
9. The method as claimed in claim 1, wherein the cell expresses both of TLR-4 and TLR-9.
- Markovic et al., Graphene quantum dots as autophagy-inducing photodynamic agents, 2012, Biomaterials, 33, 7084-7092.
- Zabirnyk, nanoparticles as a novel class of autophagy activators, 2007, autophagy, online, 1554-8635.
- Guan-Yu Chen et al., “Simultaneous induction of autophagy and toll-like recepto signaling pathways by graphine oxide”, Biomaterials 33 (Jun. 15, 2012) pp. 6559-6569.
Type: Grant
Filed: Feb 13, 2015
Date of Patent: Aug 23, 2016
Patent Publication Number: 20150157659
Assignee: National Tsing Hua University (Hsinchu)
Inventors: Yu-Chen Hu (Hsinchu), Guan-Yu Chen (Hsinchu), Hsing-Yu Tuan (Hsinchu)
Primary Examiner: Mina Haghighatian
Assistant Examiner: Luke Karpinski
Application Number: 14/622,457
International Classification: A61K 33/00 (20060101); A61K 31/194 (20060101); A61K 9/00 (20060101); A61K 9/14 (20060101);